Synthesis of a [18F]fluorobenzothiazole as potential amyloid imaging agent

Article abstract of DOI:10.1002/jlcr.1476

This study describes the synthesis of a fluoroethylated derivative of [N-methyl-¹¹C]2-(4′-methylaminophenyl)-6-hydroxybenzothiazole ([¹¹C]6-OH-BTA-1; Pittsburgh Compound B (PIB)), an already established amyloid imaging agent. The [¹¹C]methylamino group of [¹¹C]6-OH-BTA-1 was formally replaced by a fluoroethyl group in a cold synthesis via N-alkylation of N-Boc-2-(4′-aminophenyl)-6- (methoxyethoxymethoxy)benzothiazole with fluoroethyl tosylate. Subsequent deprotection gave the target compound 2-[4′-(2-fluoroethyl)aminophenyl]-6- hydroxybenzothiazole (FBTA). In a radioligand competition assay on aggregated synthetic amyloid fibrils using N-[³H-methyl]6-OH-BTA-1, 100 nM FBTA inhibited binding with 93 ± 1 and 83 ± 1% efficiency for Aβ_1-40 and Aβ_1-42, respectively. For the radiosynthesis a precursor carrying a tosylethyl moiety was prepared allowing the introduction of [¹⁸F]fluoride via nucleophilic substitution with [¹⁸F]tetra-n-butyl-ammonium fluoride (TBAF). Subsequent removal of all protecting groups was performed in a one-pot procedure followed by semi-preparative HPLC, delivering the target compound [¹⁸F]FBTA in good radiochemical yield of 21% on average and radiochemical purity of ≥98% at EOS. In vitro autoradiography on human postmortem AO brain tissue slices showed intense cortical binding of [¹⁸F]FBTA (1 nM), which was displaced in presence of 6-OH-BTA-1 (1 μM). Brain up-take was evaluated in wild-type (wt) mice with microPET imaging. Based on these results, [ ¹⁸F]FBTA appears to be a suitable candidate tracer for amyloid imaging in humans. Copyright

Full text of DOI:10.1002/jlcr.1476

Research Article
Received 2 July 2007,
Revised 4 October 2007,
Accepted 8 October 2007
Published online 1 February 2008 in Wiley Interscience
(www.interscience.wiley.com) DOI: 10.1002/jlcr.1476
Synthesis of a [18F]fluorobenzothiazole as
potential amyloid imaging agent
Ursula Berndt,a,b Christian Stanetty,a Thomas Wanek,b Claudia Kuntner,b
Johann Stanek,b Michael Berger,c Martin Bauer,d Gjermund Henriksen,e
e
Hans-Jurgen Wester, Herbert Kvaternik, Peter Angelberger,
b
b
¨
Ã
and Christian Noea
This study describes the synthesis of
a
fluoroethylated derivative of [N-methyl-11C]2-(40-methylaminophenyl)-6-
hydroxybenzothiazole ([11C]6-OH-BTA-1; Pittsburgh Compound B (PIB)), an already established amyloid imaging agent.
The [11C]methylamino group of [11C]6-OH-BTA-1 was formally replaced by a fluoroethyl group in a cold synthesis via
N-alkylation of N-Boc-2-(40-aminophenyl)-6-(methoxyethoxymethoxy)benzothiazole with fluoroethyl tosylate. Subsequent
deprotection gave the target compound 2-[40-(2-fluoroethyl)aminophenyl]-6-hydroxybenzothiazole (FBTA). In a radioligand
competition assay on aggregated synthetic amyloid fibrils using N-[3H-methyl]6-OH-BTA-1, 100 nM FBTA inhibited binding
with 93 1 and 83 1% efficiency for Ab1–40 and Ab1–42, respectively. For the radiosynthesis a precursor carrying a
tosylethyl moiety was prepared allowing the introduction of [18F]fluoride via nucleophilic substitution with [18F]tetra-n-
butyl-ammonium fluoride (TBAF). Subsequent removal of all protecting groups was performed in a one-pot procedure
followed by semi-preparative HPLC, delivering the target compound [18F]FBTA in good radiochemical yield of 21% on
average and radiochemical purity of X98% at EOS. In vitro autoradiography on human postmortem AD brain tissue slices
showed intense cortical binding of [18F]FBTA (1 nM), which was displaced in presence of 6-OH-BTA-1 (1 lM). Brain up-take
was evaluated in wild-type (wt) mice with microPET imaging. Based on these results, [18F]FBTA appears to be a suitable
candidate tracer for amyloid imaging in humans.
Keywords: Alzheimer’s disease; PET; [18F] FBTA; amyloid; [11C] BTA
similar [18F]labelled analogs of [11C]6-OH-BTA-1 are shown in
Figure 2 (compounds I–IV).
Introduction
Amyloid b (Ab) plaques and neurofibrillary tangles (NFTs)
detected in human brain postmortem are the pathological
features of Alzheimer’s disease (AD).1–3 Ab plaques are mainly
composed of aggregated Ab peptide fibrils, while NFTs consist
primarily of filaments of hyperphosphorylated tau proteins.
Clinical guidelines for AD diagnosis currently lack sensitivity and
specificity.4 Thus, the gold standard for definite diagnosis of AD
is the postmortem neuropathological examination of the brain.5
In vivo imaging of Ab plaques would greatly improve diagnosis
and therapy monitoring of AD. Based on the structure of the
histological amyloid staining reagents Congo Red and Thioflavin
T, several radiolabelled Ab-specific imaging agents have been
developed.6 [18F]labelled benzothiazole,7 thiophene,8 stilbene,9
imidazopyridine10,11 and naphtylidene12 derivatives have been
evaluated for the use as PET tracers. Up to the present
[18F]FDDNP,13,14 [11C]SB-13,15,16 [11C]BF-227,17 [18F]AV-1/ZK18
and [11C]6-OH-BTA-17,19 have been evaluated as Ab tracers in
clinical trials and are shown in Figure 1.
Among them IV ( 5 FBTA), which was first mentioned by
Mathis et al.20, has the closest structural analogy to 6-OH-BTA-1
and was chosen as our target compound22 to be evaluated as
potential PET tracer for Ab imaging. The synthesis of the cold
standard material together with its confirmation as potent Ab
radioligand in a competition assay on aggregated synthetic
amyloid fibrils using N-[3H-methyl]6-OH-BTA-1 is reported. In the
radiosynthesis, [18F]FBTA was prepared by direct introduction of
[18F]fluoride via nucleophilic substitution with [18F]tetra-n-butyl-
aDepartment of Medicinal and Pharmaceutical Chemistry, University of Vienna,
Vienna, Austria
bAustrian Research Centers GmbH-ARC, Radiation Safety and Applications,
Seibersdorf, Austria
cCenter of Brain Research, Medical University of Vienna, Vienna, Austria
dDepartment of Clinical Pharmacology, Medical University of Vienna, Vienna,
Austria
While the short half-life of the [11C]radionuclide requires
radiosynthesis in house, [18F]labelled compounds allow centra-
lized production and distribution to a larger community.
Therefore, [18F]derivatives of [11C]6-OH-BTA-1, which was the
first Ab PET tracer used in humans and is now frequently used,
have been developed.20,21 A selection of the some structurally
e
Department of Nuclear Medicine, Technische Universitat Munchen, Munich,
¨
¨
Germany
*Correspondence to: Christian Noe, Department of Medicinal and Pharmaceu-
tical Chemistry, University of Vienna, Vienna, Austria.
E-mail: christian.noe@univie.ac.at
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U. Berndt et al.
Figure 1. Clinical evaluated PET tracers for amyloid-b imaging: [ [
11C]6-OH-BTA-1: [N-methyl-11C]2-(40-methylaminophenyl)-6-hydroxybenzo-thiazole; 11C]SB-13:
[N-methyl-11C]4-N-methylamino-40-hydroxystilbene; [18F]FDDNP: [18F](2-(1-[6-[(2-[18F]fluoroethyl)(methyl)amino]-2-naphthyl]ethylidene)); [11C]BF-227: [N-methyl-11C]2-(2-
[2-dimethylaminothiazol-5-yl]ethenyl)-6-(2-[fluoro]ethoxy)benzoxazole.
Figure 2.
A selection of [ [ [ [
18F]labelled 6-OH-BTA-1 analogs: I: 18F]2-(30fluoro-40-methylaminophenyl)-6-hydroxybenzothiazole; II: 18F]6-OH-BTA-fp-1: 18F]2-[40-(3-
fluoropropyl)aminophenyl]-6-hydroxybenzothiazole; III: [18F]N-methyl-2-[40-(2-fluoroethyl)aminophenyl]-6-hydroxybenzothiazole; IV: [18F]FBTA: [18F]2-[40-(2-fluoroethyl)
aminophenyl]-6-hydroxybenzothiazole.
ammonium fluoride (TBAF). The kinetics of [18F]FBTA in the brain the MEM group in the HPLC-based monitoring of the one-pot
of wild-type (wt) mice were studied by means of microPET deprotection in the planned radiosynthesis. In general, the
imaging and the specificity of its binding was tested with deprotection of 5 was accomplished in a one-pot procedure
autoradiography using human AD brain slices with and without with TFA/DCM at reflux temperature to give 7 ( 5 FBTA) in high
the addition of a molar excess of unlabelled 6-OH-BTA-1.
and reproducible yield.
In addition to the synthesis of FBTA, we intended to
synthesize the structurally closest derivative of 6-OH-BTA-1,
the N-fluoromethyl derivative 10. In this approach, we
succeeded in the analogous alkylation of carbamate 4 with
fluoromethyl tosylate27 giving compound 8. Unfortunately,
Results and discussion
Chemistry
Based on protocols from the literature,7 2-(4-nitrophenyl)-6- compound 8 turned out to be unstable and degraded to
hydroxybenzothiazole 1 was synthesized in four steps starting hydroxymethyl derivative 9 upon purification on silica gel and
from 4-nitrobenzoic acid and p-anisidine. The phenolic moiety under storage in the refrigerator. Attempted acidic deprotection
was protected as the methoxyethoxymethyl (MEM) ether23 to to compound 10 was unsuccessful as well (see Scheme 3).
give 2. Reduction of the nitro group7 with NaBH4 in ethanol lead
These results were not completely unexpected since there is
to aniline 3, which was protected with a Boc group under no satisfying literature precedence for the synthesis of a
standard conditions24,25 to give compound 4 (Scheme 1), in secondary fluoromethylaniline, neither in cold nor hot synthesis
order to prevent dialkylation in the labelling step. Compound 4 although several patents28,29 quote structures of cold fluoro-
is the key intermediate for the synthesis of cold reference methylated anilines - unfortunately not including any experi-
material and for the radiosynthesis of [18F]FBTA.
mental procedures or analytical data. Zheng and Berridge,30
The alkylation of 4 was achieved in dry dimethylfomamide have reported comprehensively on the alkylation of diphenyl-
(DMF) with NaH as base at 601C using fluoroethyltosylate26 as amine with [18F]fluoroiodomethane, among other tertiary
alkylating agent giving compound 5 as stable and storable fluoromethyldialkylamines. Examples of N-fluoromethylated
material in high yield (Scheme 2).
nitrogen containing heterocycles that have been used as PET
The lower stability of the Boc group compared with the MEM tracers in other regards are described elsewhere.31,32 Encour-
group allowed selective cleavage with 5% trifluoroacetic acid aged by the results of Zheng and Berridge,30 we attempted
(TFA) in dichloromethane (DCM) at room temperature (rt) alkylation of the unprotected aniline 3 with fluoromethyl
yielding fluoroethylamine 6. Compound 6 was needed as tosylate in various solvents like chloroform, DMF or acetone
reference material for the detection of incomplete cleavage of with different bases, which all remained unsuccessful.
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U. Berndt et al.
Scheme 1
Scheme 2
Scheme 3
was analysed by analytical HPLC. Comparison with cold standard
material 5 proved successful [18F]fluorination of the precursor.
Radiosynthesis
Although there is literature33 covering the [18F]fluoroethylation Cleavage of the Boc group and the MEM group was carried out
of other substrates with [18F]fluoroethyl tosylate we decided to in a one-pot procedure with HCl/MeOH. Before injection onto
synthesize precursor 11 carrying a tosylethyl group, thus the semipreparative HPLC, the reaction mixture was neutralized
allowing nucleophilic introduction of [18F]fluoride. In this way, with NH4OH and diluted with acetonitrile to redissolve
we circumvent the laborious two-step radiosynthesis using precipitated material, which is crucial to achieve high isolated
[18F]fluoroethyl tosylate as an alkylating agent (see Scheme 4).
yield. The high lipophilicity of the target compound did not
The synthesis of the precursor 11 was accomplished by the allow the use of a physiologic eluent in the HPLC purification,
alkylation of intermediate 4 with ethylene bis-tosylate.34 The which was complicating the work up procedure; [18F]FBTA was
alkylation was carried out under similar conditions as the eluted with acetonitrile/TEAP-buffer (Figure 3, trace A).
alkylation of compound 4 with fluoromethyl and fluoroethyl
The product peak was collected and diluted with water. This
tosylate, but had to be performed at lower temperature of 401C solution was adsorbed on a C18-cartridge and subsequently
to avoid the formation of by-products 12 and 13.35 At higher washed with water. The product was eluted with ethanol and
temperatures, cyclization of precursor 11 under the loss of the transferred into an injectable solution. Radiochemical purity was
tert-butyl group yielding cyclic carbamate 12 was observed. The greater than 98% at the end of synthesis (EOS) and was still
same cyclization was observed when the precursor was stored greater than 95% after 6 h as measured by analytical HPLC
at rt. By-product 13 was formed from ethylene bis-tosylate at (Figure 3, trace B). The chemical identity was confirmed by
elevated temperature.
co-injection with the cold standard compound (Figure 3, trace
For the radiosynthesis, [18F]fluoride was generated from C). (Retention time of FBTA was 10.0 min versus 9.9 min of
H2[18O]-enriched water via proton bombardment. [18F]fluoride [18F]FBTA because the sample passed the radioactivity detector
was adsorbed on an anion exchange cartridge as described in first and UV detector second in the used HPLC system.) The
the literature36 and eluted with tetra-n-butyl-ammonium product contained no detectable amount of precursor and
hydrogen carbonate to give [18F]TBAF. Precursor 11 was 0.7 nmol (mean) FBTA per mCi at EOS. The radiosynthesis of
transformed into the [18F]fluoroethyl compound [18F]5 via [18F]FBTA was performed in a PC-controlled synthesizer module
nucleophilic substitution with [18F]TBAF37 in dry acetonitrile at that had been adapted to perform all steps leading to [18F]FBTA
elevated temperature. A crude sample of the reaction mixture automatically. The title compound was obtained in a radio-
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U. Berndt et al.
Scheme 4
Figure 3. Isolation and analysis of [18F]FBTA: trace A: semipreparative HPLC of the reaction mixture: radioactivity detector; [18F]fluoride retention time 3.2 min (58.4%),
18F]FBTA retention time 9.9 min (41.6%). Trace B: analytical HPLC of the end product: radioactivity detector; [18F]fluoride retention time 2.8 min (1.7%); [18F]FBTA retention
time 9.9 min (98.3%). Trace C: analytical HPLC of FBTA standard: UV detector; retention time 10.0 min.
[
chemical yield of 2175% (n 5 13) with a mean specific activity This is less compared with [11C]6-OH-BTA-17 but is still in a
of 2.571.2 mCi/nmol (n 5 7) at EOS. [18F]FBTA was shown to be useful range for use as a PET tracer since [18F]FBTA allows a
stable in vitro and is available for routine synthesis.
longer time of measurement.
Binding affinities of FBTA for aggregated synthetic amyloid
fibrils (Ab1–40 and Ab1–42) were determined in a radioligand
competition assay using N-[3H-methyl]6-OH-BTA-1 according to
literature.38 At a concentration of 100 nM, FBTA inhibited [3H]6-
OH-BTA-1 binding with 9371 and 8371% efficiency for Ab1–40
and Ab1–42, respectively. In comparison, cold 6-OH-BTA-1 tested
in an identical manner, resulted in 7272 and 7073% inhibition
of binding for Ab1–40 and Ab1–42, respectively.
In vitro and in vivo evaluation
The kinetics of [18F]FBTA in brains of wt mice were studied by
means of microPET imaging. Following i.v. administration of
[18F]FBTA, peak radioactivity uptake in mouse brain was in the
order of about 0.1% ID kg/g. We analysed frontal cortex as a
region where plaque load would be expected in human AD
patients and cerebellum as a region which has been shown to
be devoid of plaque load. In Figure 4, dose-normalized [18F]FBTA
time–activity curves for the frontal cortex and the cerebellum
are shown. The ratio of the radioactivity concentration in frontal
cortex at 2 min to that at 30 min after tracer injection was 3.5.
Further, binding of [18F]FBTA to Ab plaques was also proven
by autoradiography of human congophilic AD brain slices.
Incubation39 and washing40 of the slices were done according to
literature with minor modifications. Intense cortical binding was
observed (Figure 5(a)). In a displacement experiment done in
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U. Berndt et al.
Figure 4. Mean TACs of three wt mice in the frontal cortex and in the cerebellum.
model 200 was used. The semipreparative HPLC was equipped
with a WellChrom Filter-Photometer K-2001 from Knaur and a
Geiger–Mu¨ller tube as a radioactivity detector. UV detection was
done at 254 nm. The radiosynthesis was performed in a PC-
controlled TRACERlab FX-FDG Synthesiser Module from GE
Healthcare, which had been adapted for the synthesis of
[18F]FBTA. The system was operated with the GINA StarTM 4.0
software.
Figure 5. Specific Binding (a: ꢀ1 nM [18F]FBTA) and selective displacement (b:
ꢀ1 nM
18F]FBTA11 mM 6-OH-BTA-1) of 18F]FBTA to Ab plaques shown by
autoradiography using postmortem human brain tissue sections.
[
[
Melting points were measured on a Leica Galen III instrument
and are uncorrected. 1H-NMR spectra were recorded on a
Bruker-Spectrospin 200 (200 and 50 MHz, respectively) spectro-
meter or a Varian Unity spectrometer (500 and 125 MHz,
respectively). HRMS were obtained with a Finnigan MAT 900
(ESI) or a Finnigan MAT 8230 (EI). Elemental analysis was
performed on a 2400 CHN Elemental Analyzer from Perkin
Elmer.
parallel selective displacement of [18F]FBTA by unlabelled 6-OH-
BTA-1 can clearly be seen (Figure 5(b)).
Conclusion
Radiosynthesis of a [18F]labelled analog of [11C]6-OH-BTA-1 was
successfully accomplished. The [11C]methyl group was formally
replaced by a [18F]fluoroethyl group. Cold reference material
FBTA was obtained by high-yielding alkylation of Boc aniline 4
with fluoroethyltosylate and subsequent deprotection. In the
hot synthesis the lower yielding alkylation with ethylene
bistosylate giving tosylethyl precursor 11 was accepted to allow
one-step nucleophilic introduction of [18F]fluoride with
[18F]TBAF followed by one-pot deprotection giving [18F]FBTA.
A detailed discussion of the problems encountered in the
attempted synthesis of fluoromethyl derivative 10 is included in
this report. Acceptable peak brain uptake at 2 min p.i. and 2 min/
30 min washout ratios in brains of wt mice indicate that
[18F]FBTA might be a useful Ab tracer in humans. Binding affinity
of FBTA for Ab1–40 and Ab1–42 fibrils, as determined by an
inhibition assay using N-[3H-methyl]6-OH-BTA-1, was shown to
be higher than that of 6-OH-BTA-1. Autoradiography of human
AD brain slices showed that [18F]FBTA binds specific and in a
displaceable manner to the same binding sites as 6-OH-BTA-1.
Synthetic chemistry
2-(40-Nitrophenyl)-6-(methoxyethoxymethoxy)benzothiazole (2):
2-(40-Nitrophenyl)-6-hydroxybenzothiazole 1 (2.68 g, 9.8 mmol)
was dissolved in DCM (100 ml) before N-ethyl-di-isopropylamine
(Huenig’s base) (4.8 ml, 41.8 mmol) and MEM-Cl (7.3 ml,
34.3 mmol) were added successively. The solution was stirred
at rt for 49 h. The reaction mixture was washed with 0.05 N HCl
and 0.05 N NaOH, dried over Na2SO4 and evaporated. The crude
product was purified by flash column chromatography (SiO2;
LP:EtOAc 5 4:1) to give 2.39 g (68%) of the pure product as
yellow solid.
1H-NMR (200 MHz, CDCl3) d 5 8.35 (d, J 5 9.0 Hz, 2H), 8.22 (d,
J 5 8.8 Hz, 2H), 8.01 (d, J 5 9.0 Hz, 1H), 7.65 (d, J 5 2.4 Hz, 1H),
7.25 (dd, J 5 2.7, J 5 8.6, 1H), 5.37 (s, 2H), 3.90–3.86 (m, 2H),
3.61–3.57 (m, 3H), 3.39 (s, 3H). 13C-NMR (50 MHz, CDCl3)
d 5 162.9 (C), 156.0 (C), 149.3 (C), 148.7 (C), 139.2 (C), 136.8 (C),
127.8 (CH), 124.4 (CH), 124.2 (CH), 117.9 (CH), 107.4 (CH), 93.9
(CH2), 71.5 (CH2), 67.9 (CH2), 59.0 (CH3). Calculated for
Experimental
All starting materials and reagents were purchased from C17H16N2O5S: C 55.66%; H 4.48%; N 7.77%; S 8.90%; found:
commercial sources and used without further purification. C 56.32%; H 4.34%; N 7.79%; S 8.71%. Melting point: 144–1461C
Analytical HPLC and semipreparative HPLC were performed (analytical sample was recrystallized from EtOAc).
with an Agilent ChemStation 1100 System, which was
2-(40-Aminophenyl)-6-(methoxyethoxymethoxy)benzothiazole (3):
equipped with a quaternary gradient pump and was operated To a mixture of 2-(40-nitrophenyl)-6-(methoxyethoxymethoxy)-
with the designated software. In analytical HPLC a multi- benzothiazole 2 (4.83 g, 13.40 mmol) and Cu(OAc)2 (2.43 mg,
wavelength UV detector and a Gabi Star scintillation detector 13.40 mmol) in anhydrous ethanol (160 ml), NaBH4 (17.74 g,
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U. Berndt et al.
469.0 mmol) was added portionwise and the reaction mixture
2-[40-(2-Fluoroethyl)aminophenyl]-6-(methoxyethoxymethoxy)-
was stirred at rt for 20 h. The solvent was evaporated and the benzothiazole (6): TFA (2.5 ml) was added to a solution of 5
residue was taken up in water and extracted with EtOAc. The (150 mg, 0.32 mmol) in DCM (47.5 ml). The solution was stirred at
extracts were combined, dried over Na2SO4 and evaporated. rt for 1 h. The solvent was evaporated. The residue was dissolved
The crude product was purified by flash column chromatogra- in DCM, washed with water and 2N NaOH, dried over Na2SO4
phy (SiO2; LP:EtOAc 5 1:2) to give 2.76 g (62%) of the pure and evaporated. The crude product was purified by flash column
product as yellow solid.
chromatography (SiO2; LP:MTBE 5 3:1) to give 73 mg (62%) of
1H-NMR (200 MHz, CDCl3) d 5 7.88 (d, J 5 6.1 Hz, 1H), 7.84 (d, the pure product as yellow solid.
J 5 5.9 Hz, 2H), 7.55 (d, J 5 2.4 Hz, 1H), 7.14 (dd, J 5 2.5 Hz,
1H-NMR (200 MHz, MeOD) d 5 7.81 (d, J 5 2.7 Hz, 1H), 7.76 (d,
J 5 8.8 Hz, 1H), 6.71 (d, J 5 8.5 Hz, 2H), 5.32 (s, 2H), 3.89–3.84 (m, J 5 2.9 Hz, 2H), 7.60 (d, J 5 2.4 Hz, 1H), 7.17 (dd, J 5 2.5 Hz,
2H), 3.60–3.55 (m, 2H), 3.38 (s, 3H). 13C-NMR (50 MHz, CDCl3) J 5 8.8 Hz), 6.73 (d, J 5 8.8 Hz, 2H), 5.32 (s, 2H), 4.59 (dt,
d 5 166.8 (C), 154.8 (C), 149.3 (C), 149.0 (C), 135.5 (C), 128.9 (CH), J 5 47.6 Hz, J 5 5.0 Hz, 2H), 3.86–3.82 (m, 2H), 3.60–3.53 (m,
123.8 (C), 122.8 (CH), 116.7 (CH), 114.8 (CH), 107.7 (C), 94.0 (CH2), 3H), 3.43 (t, J 5 5.1 Hz, 1H), 3.33 (s, 3H). 13C-NMR (50 MHz, CDCl3)
71.6 (CH2), 67.7 (CH2), 59.0 (CH3). Calculated for C17H18N2O3S: d 5 156.4 (C), 152.3 (C), 150.3 (C), 136.4 (C), 129.8 (CH), 123.1
C 61.80%, H 5.49%. N 8.48%, S 9.70%; found: C 61.55%; H 5.58; (CH), 122.7 (C), 118.0 (CH), 113.3 (CH), 109.1 (CH), 95.2 (CH2), 83.4
N 8.48%; S 9.39%. Melting point: 145–1471C (analytical sample (d, J 5 167.6 Hz, CH2), 72.8 (CH2), 68.9 (CH), 59.1 (CH3), 44.5
was recrystallized from MeOH).
N-Boc-2-(40-aminophenyl)-6-(methoxyethoxymethoxy)benzo-
(N–CH2–CH2–F, d, J 5 21.2). Calculated for C19H21FN2O3S:
C 60.62%, H 5.62%, N 7.44%, S 8.52%, F 5.05%; found:
thiazole (4): A mixture of 3 (2.00 g, 6.05 mmol) and di-tert-butyl C 60.53%, H 5.65%, N 7.45%, S 8.34%, F 5.31%. Melting point:
dicarbonate (3.02 g, 13.84 mmol) in THF (10 ml) was heated to 84–851C (analytical sample was recrystallized from MeOH).
reflux for 42 h. The mixture was poured on water and extracted
2-[40-(2-Fluoroethyl)aminophenyl]-6-hydroxybenzothiazole (7):
with DCM. The combined organic layers were washed with TFA (5 ml) was added to a solution of 5 (851 mg, 1.79 mmol) in
water, dried over Na2SO4 and concentrated. The crude product DCM (30 ml) and the solution was stirred at reflux for 1 h. The
was purified by flash column chromatography (SiO2; LP: reaction mixture was allowed to cool to rt and a saturated
Et2O 5 1:2) to give 2.53 g (97%) of the pure product as yellow solution of NaHCO3 was added until the aqueous layer reached
solid.
a pH of 8. The product was extracted with MTBE. The combined
1H-NMR (500 MHz, CDCl3) d 5 7.97 (d, J 5 8.5 Hz, 2H), 7.92 (d, organic layers were dried over Na2SO4 and evaporated. The
J 5 9.1 Hz, 1H), 7.58 (d, J 5 2.2 Hz, 1H), 7.48 (d, J 5 8.5 Hz, 2H), crude product was purified by flash column chromatography
7.17 (dd, J 5 2.4 Hz, J 5 9.0 Hz, 1H), 6.71 (s, 1 H, NH), 5.33 (s, 2H), (SiO2; LP:MTBE 5 1:1) to give 287 mg (56%) of the pure product
3.89–3.86 (m, 2H), 3.59–3.57 (m, 2H), 3.38 (s, 3H), 1.53 (s, 9H). 13C- as yellow solid.
NMR (125 MHz, CDCl3) d 5 166.0 (C), 155.1 (C), 152.3 (C), 149.3
1H-NMR (500 MHz, CD3CN) d 5 7.80 (d, J 5 8.8 Hz), 7.71 (d,
(C), 140. 73 (C), 135.9 (C), 128.2 (CH), 123.3 (CH), 118.3 (CH), 116.9 J 5 8.8 Hz, 1H), 7.32 (d, J 5 2.1 Hz, 1H), 6.94 (dd, J 5 2.5 Hz,
(CH) 107.6 (CH), 94.0 (CH2), 81.1 (C), 71.6 (CH2), 67.8 (CH2) 59.0 J 5 8.5 Hz, 1H), 6.72 (d, J 5 8.8 Hz, 2H), 4.58 (dt, d, J 5 47.4 Hz,
(CH3), 28.3 (CH3). Calculated for C22H26N2O5S: C 61.38%; H 6.09%; J 5 5.0 Hz, 2H), 3.47 (dt, d, J 5 26.9 Hz, J 5 5.0 Hz, 2H). 13C-NMR
N 6.515; S 7.45%; found: C 61.05%; H 6.22%; N 6.54%; S 7.17%. (125 MHz, CD3CN) d 5 166.3 (C-2), 155.5 (C-6), 151.7 (C-40), 149.2
Melting point: 127–1291C (analytical sample was recrystallized (C-3a), 136.7 (C-7a), 129.4 (C-20, C-60), 123.6 (C-4), 123.2 (C-10),
from MeOH).
116.3 (C-5), 113.3 (C-30, C-50), 107.7 (C-7), 83.6 (d, J 5 165.4 Hz,
CH2F), 44.2 (d, J 5 19.4 Hz, CH2N). HRMS (EI) m/z calculated
N-Boc-2-[40-(2-fluoroethyl)aminophenyl]-6-(methoxyethoxy-
methoxy)benzothiazole (5): NaH (27 mg, 1.28 mmol) was for C15H13FN2OS 288.0733; found: [M]1 288.0736. Melting
dissolved in dry DMF (3 ml) with external ice/salt cooling. point: 194–1961C (analytical sample was recrystallized from
Boc-amine 4 (243 mg, 0.56 mmol) and fluoroethyl tosylate LP/MTBE).
(247 mg, 1.13 mmol) were added and the reaction mixture
N-Boc-2-[40-(fluoromethylamino)phenyl]-6-(methoxyethoxy-
was stirred at 601C for 3 h. It was cooled to rt, diluted with methoxy)benzothiazole (8): NaH (19 mg, 0.79 mmol) was
MTBE, quenched with water and extracted with MTBE. dissolved in dry DMF (2 ml) with external ice/salt cooling. Boc-
The combined organic layers were washed with water, amine
4 (171 mg, 0.40 mmol) and fluoromethyl tosylate
dried over Na2SO4 and evaporated to give the crude product, (16.2 mg, 0.79 mmol) were added and the reaction mixture
which was purified by flash column chromatography (SiO2; was stirred at 601C for 3 h. It was cooled to rt, diluted with
LP:Et2O 5 1:1) to give 247 mg (92%) of the pure product as MTBE, quenched with water and extracted with MTBE. The
yellow solid.
combined organic layers were washed with water, dried over
1H-NMR (200 MHz, CDCl3) d 5 8.01 (d, J 5 8.6 Hz, 2H), 7.94 (d, Na2SO4 and evaporated to give the crude product, which was
J 5 9.0 Hz, 1H), 7.60 (d, J 5 2.4 Hz, 1H), 7.36 (d, J 5 8.6 Hz, 2H), purified by flash column chromatography (SiO2; LP:Et2O 5 1:1)
7.19 (dd, J 5 2.5 Hz, J 5 9.0 Hz, 1H), 5.34 (s, 2H), 4.62 (dt, to give 38 mg (21%) of the pure target compound as
J 5 47.4 Hz, J 5 4.9 Hz, 2H), 4.01 (t, J 5 4.99 Hz, 1H), 3.91–3.85 yellow oil.
(m, 3H), 3.60–3.56 (m, 2H), 3.38 (s, 3H), 1.45 (s, 9H). 13C-NMR
1H-NMR (200 MHz, CDCl3) d 5 8.03 (d, J 5 8.5 Hz, 2H), 7.95 (d,
(50 MHz, CDCl3) d 5 165.5 (C), 155.3 (C), 154.1 (C), 149.4 (C), J 5 9.0 Hz, 1H), 7.60 (d, J 5 2.4 Hz, 1H), 7.42 (d, J 5 8.6 Hz, 2H),
144.8, 136.2 (C), 131.3 (C), 127.7 (CH), 127.3 (CH), 123.6 (CH), 7.19 (dd, J 5 2.5 Hz, J 5 8.9 Hz), 5.68 (d, J 5 54.8 Hz, 2H), 5.34
(CH), 117.1 (CH), 107.6 (CH), 94.0 (CH2), 81.6 (d, J 5 169.8 Hz, (s, 2H), 3.89–3.85 (m, 2H), 3.60–3.55 (m, 2H), 3.38 (s, 3H), 1.49 (s,
CH2), 81.2 (C), 76.4 (CH2), 71.6 (CH2), 59.0 (CH3), 50.6 (d, 9H). 13C-NMR (50 MHz, CDCl3) d 5 165.2 (C), 155.3 (C), 153.3 (C),
J 5 20.6 Hz, CH2), 28.3 (CH3). Calculated for C24H29FN2O5S: 149.4 (C), 143.3/143.2 (C), 136.3 (C), 132.1 (C), 127.7 (CH), 126.63/
C 60.49%, H 6.13%, N 5.88%, S 6.73%; found: C 6.34%, H 126.60 (CH), 123.7 (C), 117.1 (C), 107.6 (C), 93.9 (CH2), 88.5 (d,
6.28%, N 5.91%, S 6.55%. Melting point: 70–721C (analytical J 5 199.1 Hz, CH2), 82.6 (C), 71.6 (CH2), 67.8 (CH2), 59.0 (CH3), 28.1
sample was recrystallized from Et2O).
(CH3).
www.jlcr.org
Copyright r 2008 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2008, 51 137–145

U. Berndt et al.
N-Boc-2-[40-(hydroxymethylamino)phenyl]-6-(methoxyethoxy- exchange cartridge (SEP-PAK light, Waters Accell Plus QMA,
methoxy)benzothiazole (9): Obtained as a degradation product USA). Elution of the activity was achieved with 0.5 ml of tetra-n-
from 8: 1H-NMR (500 MHz, DMSO) d 5 8.01 (d, J 5 8.5 Hz, 2H), butyl-ammonium hydrogen carbonate (38 mmol, pHꢀ8) in
7.96 (d, J 5 9.1 Hz, 1H), 7.80 (d, J 5 2.2 Hz, 1H), 7.49 (d, J 5 8.5 Hz, water. Azeotropic drying was repeatedly performed with
2H), 7.22 (dd, J 5 2.4 Hz, J 5 9.0 Hz, 1H), 6.21 (t, J 5 7.0 Hz, 1H), acetonitrile in a glassy carbon (SigradursG) reaction vessel.
5.35 (s, 2H), 5.00 (d, J 5 7.0 Hz, 2H), 3.77–3.75 (m, 2H), 3.49–3.47 The precursor 11 (5 mg, 16 mmol) was dissolved in dry
(m, 2H), 3.22 (s, 3H), 1.43 (s, 9H). 13C-NMR (125 MHz, DMSO) acetonitrile (1 ml), added to the dry n.c.a. [18F]TBAF and the
d 5 164.8 (C-2), 154.8 (C-6), 152.6 (COON), 148.8 (C-3a) 144.5 (C- mixture was heated to 901C for 5 min to achieve nucleophilic
40), 135.7 (C-7a), 130.0 (C-10), 127.1 (C-20, C-40), 126.5 (C-30, C-50), substitution of the tosyl group.
123.4 (C-4), 117.2 (C-5), 108.2 (C-7), 93.4 (O–CH2–O), 80.4 (t-Bu),
72.5 (N–CH2–O), 71.0 (O–CH2–CH2–O), 67.5 (O–CH2–CH2–O), 58.0 HPLC to determine percent incorporation into [18F]5. Retention
(OCH3), 27.9 (t-Bu-CH3). time (reaction intermediate): 23.9 min. Column: Hamilton PRP-1,
A sample of the reaction mixture was analysed by analytical
N-Boc-2-[40-(2-tosylethyl)aminophenyl]-6-(methoxyethoxy- 10 mm 290 ꢁ 4 mm analytical column (solvent A: 0.2% triethy-
methoxy)benzothiazole (11): NaH (7 mg, 0.29 mmol) was lammonium phosphate buffer pH 7.2, solvent B: acetonitrile;
dissolved in dry DMF (0.8 ml) and Boc-amine 4 (124 mg, gradient: 1–10 min 50% B, 10–24 min 50–100% B, 24–30 min
0.26 mmol) was added at 01C. This solution was added dropwise 100% B; flow 1 ml/min).
to a solution of ethylene bis-tosylate (213 mg, 58 mmol)
[18F]2-[40-(2-fluoroethyl)aminophenyl]-6-hydroxybenzothiazole
in dry DMF (0.8 ml) at 401C. Upon complete addition, ([18F]FBTA 5 [18F]7): After the nucleophilic substitution, the
the reaction mixture was stirred at 401C for another reaction mixture was evaporated to dryness by an inert gas
10 min before it was cooled to rt, diluted with MTBE (3 ml), stream for 5 min, maintaining the temperature at 601C. After
quenched with water (5 ml) and finally extracted with MTBE. cooling to about 451C a solution of MeOH/HCl (1 ml, 2/1 MeOH/
The combined organic layers were washed with water and concentrated HCl) was added and the mixture was heated for 5
brine, dried over Na2SO4 and evaporated. The crude product min at 901C. The reaction mixture was cooled down to 401C,
was purified by flash column chromatography (SiO2; neutralized by adding 2 ml of NH4OH (water/concentrated
LP:MTBE 5 1:1) to give 64 mg (35%) of pure target compound NH4OH 5 1/1) and diluted with 2 ml of acetonitrile. The product
as white solid.
was purified by semipreparative HPLC using a Hamilton PRP-1
1H-NMR (500 MHz, CDCl3) d 5 7.96–7.94 (m, 3H), 7.72 (d, 10 mm 250 ꢁ 8 mm semipreparative column eluted with 60%
J 5 8.2 Hz, 2H), 7.62 (d, J 5 2.1 Hz, 1H), 7.30 (d, J 5 8.2 Hz, 2H), acetonitrile/40% 0.2% triethylammonium phosphate buffer pH
7.23 (d, J 5 8.5 Hz, 2H), 7.20 (dd, J 5 2.2 Hz, J 5 8.8 Hz, 1H), 5.35 7.2 (flow 3 ml/min). The [18F]FBTA peak eluted at about 10 min.
(s, 2H), 4.24 (t, J 5 5.4 Hz, 2H), 3.91 (t, J 5 5.4 Hz, 2H), 3.89–3.87 The fraction containing [18F]FBTA was diluted with 80 ml of
(m, 2H), 3.60–3.58 (m, 2H), 3.39 (s, 3H), 1.41 (s, 9H). 13C-NMR water and adsorbed on a Waters C18 SepPak Plus cartridge. The
(125 MHz, CDCl3) d 5 165.3 (C), 155.3 (C), 153.9 (C), 144.9 (C), C18 SepPak was washed with 10 ml of water and the product
144.3 (C), 136.2 (C), 131.4 (C), 129.9 (CH), 127.9 (CH), 127.7 (CH), was eluted with 1 ml of anhydrous ethanol into a sterile vial
127.1 (CH), 123.7 (CH), 117.1 (CH), 107.6 (CH), 94.0 (CH2), 81.4 (C), followed by 9 ml of saline. Radiochemical and chemical purity
71.6 (CH2), 67.8 (CH2), 67.3 (CH2), 59.0 (CH3), 49.1 (CH2) 28.2 were 498% as determined by analytical HPLC. Retention time
(CH3), 21.6 (CH3) HRMS (ESI) m/z calculated for C31H37N2O8S2 ([18F]FBTA): 9.9 min Column: Hamilton PRP-1, 10 mm 290 ꢁ 4 mm
629.1991; found 629.2003 ([M1H]1).
analytical column (solvent A: 0.2% triethylammonium phosphate
buffer pH 7.2, solvent B: acetonitrile; gradient: 1–10 min 50% B,
2-[40-(2-Oxo-3-oxazolidinyl)phenyl]-6-(methoxyethoxymethoxy)-
benzothiazole (12): Isolated as a by-product in the synthesis of 11: 10–24 min 50–75% B, 24–30 min 75% B; flow 1 ml/min). The
1H-NMR (500 MHz, CDCl3) d 5 8.04 (d, J 5 8.7 Hz, 2H), radiochemical yield averaged 21%75
@ EOS based on
7.96 (d, J 5 8.8 Hz, 1H), 7.66 (d, J 5 8.8 Hz, 2H), 7.57 (d, [18F]fluoride, and the specific activity averaged 2.5 mCi/nmol
J 5 1.9 Hz, 1H), 7.19 (dd, J 5 2.4 Hz, J 5 9.0 Hz), 5.33 (s, 2H), 4.51 (93 GBq/mmol) @ EOS.
(t, J 5 7.7 Hz, 2H), 4.09 (t, J 5 8.0 Hz, 2H), 3.87–3.86 (m, 2H),
3.59–3.57 (m, 2H), 3.38 (s, 3H). 13C-NMR (125 MHz, CDCl3)
d 5 165.8 (C-2), 155.4 (C-6), 154.8 (COON), 148.3 (C-3a), 140.5
MicroPET imaging
(C-10, C-40), 135.6 (C-7a), 128.2 (C-20, C-60), 123.2 (C-4), 118.0 (C-30, MicroPET imaging was performed on three female wt mice C56
C-50), 117.3 (C-5), 107.5 (C-7), 93.9 (O–CH2–O), 71.5 black six (age: 6–8 weeks; weight: 21.572.7 g) using a microPET
(O–CH2–CH2–O), 67.8 (O–CH2–CH2–O), 61.3 (O–CH2), 59.0 Focus220 scanner (Siemens, Medical Solutions). The animals
(OCH3), 44.9 (N–CH2).
were kept under isoflurane anaesthesia (1.5%) and positioned
Vinyl 4-methylbenzenesulphonate (13). Isolated as a by-product on a temperature-regulated animal bed (371C) in the microPET
in the synthesis of 11: 1H-NMR (200 MHz, CDCl3) d 5 7.79 (d, scanner. [18F]FBTA was administered as an intravenous bolus
J 5 8.2 Hz, 2H), 7.36 (d, J 5 7.5 Hz, 2H), 6.60 (dd, J 5 5.8 Hz, injection (4–10 MBq in a volume of 100 ml of physiological saline/
J 5 13.5 Hz, 1H), 4.89 (dd, J 5 2.4 Hz, J 5 13.5 Hz, 1H), 4.68 (dd, ethanol, ethanol content p10% (v/v)) via the tail vein. At the
J 5 2.4 Hz, J 5 5.8 Hz), 2.45 (s, 1H). 13C-NMR (50 MHz, CDCl3) start of radiotracer injection, dynamic microPET imaging was
d 5 145.3 (C), 141.6 (CH), 132.5 (C), 129.9 (CH), 128.0 (CH), 102.7 initiated. List mode data were acquired for 60 min with an
(CH2), 21.7 (CH3).
energy window of 250–750 keV and 6 ns timing window. The
dynamic microPET image data were sorted into three-
dimensional sinograms as follows: six frames of 20 s, five frames
of 60 s, four frames of 120 s, three frames of 300 s and three
frames of 600 s. Transmission scans using a Co-57 point
source were performed on each mouse for 10 min. Dynamic
images were reconstructed by Fourier rebinning of the
Radiochemistry
[
18F]N-Boc-2-[40-(2-fluoroethyl)aminophenyl]-6-(methoxyethoxy-
methoxy)benzothiazole ([18F]5): [18F]Fluoride was separated
from the proton-irradiated H2 [18O]-enriched water on an anion
J. Label Compd. Radiopharm 2008, 51 137–145
Copyright r 2008 John Wiley & Sons, Ltd.
www.jlcr.org

U. Berndt et al.
three-dimensional sinograms followed by two-dimensional FBP
Herbert Budka from the Medical University in Vienna is
(filtered backprojection) with a ramp filter. Normalization, decay- thanked for providing human AD brain tissue.
and attenuation correction was applied to the data. MicroPET
data were analysed using a dedicated software package (PMOD,
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Acknowledgements
The authors wish to thank especially Oliver Langer from the
Medical University of Vienna (Department of Clinical Pharmacol-
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Zsebedics from the Department of Toxicology at the ARC in
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